Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase (MAP3K) that regulates multiple MAPK signaling pathways in response to mitogenic and stress stimuli. Depending on the cellular context, MLK3 can phosphorylate MKK4 or MKK7 MAPK kinases (MAP2Ks), which activate c-Jun N-terminal Kinase (JNK) to trigger cell death. Alternatively, MLK3, through a kinase-independent scaffold function, can activate B-Raf and extracellular signal-regulated kinase (ERK) signaling to promote cell proliferation. MLK3 is required for the migration and invasion of human ovarian, gastrointestinal, lung, and breast cancer cells. A requirement for MLK3 in mitogen-dependent ERK activation and colon cancer cell proliferation has been demonstrated; however its function in colon cancer cell invasion is not known. Many cancer cells have persistent, intrinsic oxidative stress and higher levels of reactive oxygen species (ROS) than normal cells. Stimuli such as pro-inflammatory cytokines, irradiation, toxins and chemotherapeutic drugs can trigger ROS production. At very high levels ROS can elicit activation of signal transduction pathways and induce apoptosis or necrotic cell death, and at low levels ROS can stimulate cell proliferation. Our preliminary findings indicate that oxidative stress strongly activates ERK1/2 signaling which promotes the phosphorylation of MLK3 in colon cancer cells. Oxidative stress also promotes an ERK-dependent increase in MLK3 protein level and a decrease in MLK3 kinase activity. Furthermore, we demonstrated that ERK could phosphorylate MLK3 in vitro. Thus, we have identified ERK as a novel, positive regulator of MLK3 protein level and a negative regulator of MLK3 kinase activity in response to oxidative stress. We postulate that the enhancement of inactive MLK3 protein in response to oxidative stress could cause persistent MLK3-dependent stimulation of ERK signaling and thereby promote cellular transformation and proliferation of colon cancer cells. The overall goal of this proposal is to define the mechanism by which oxidative stress induces ERK-dependent regulation of MLK3, and elucidate the impact of this regulation on MLK3-dependent stimulation of cellular transformation, proliferation and apoptosis in colon cancer cells. Accordingly the specific aims of the proposal are: 1) To investigate the role of ERK signaling in H202-mediated upregulation of MLK3 in colon cancer cells. 2) To determine the impact of ROS and ERK activation on MLK3 kinase activity and function, 3) To elucidate the function of MLK3 in colon cancer cell proliferation and invasion. The experiments described in this proposal will allow us to gain a thorough understanding of how oxidative stress modulates MLK3 protein level and kinase activity and how this affects MLK3-dependent activation of MAPK signaling, proliferation and invasion of colon cancer cells. Furthermore, we will examine novel strategies to deplete cellular MLK3 protein levels to inhibit colon cancer cell proliferation and invasion.